1. Field of the Invention
The invention relates to procedures and devices for fragmenting molecular ions, preferably biomolecular ions in tandem mass spectrometers.
2. Background of the Invention
Over the last decade, mass spectrometry has played an increasingly important role in the identification and characterization of biochemical compounds in research laboratories and various industries. The speed, specificity, and sensitivity of mass spectrometry make spectrometers especially attractive for requiring rapid identification and characterization of biochemical compounds. Mass spectrometric configurations are distinguished by the methods and techniques utilized for ionization and separation of the analyte molecules. The mass separation process can include techniques for ion isolation, subsequent molecular fragmentation, and mass analysis of the fragment ions. The pattern of fragmentation yields information about the structure of the analyte molecules introduced into the mass spectrometer. Increased fragmentation thus increases one's ability to distinguish one mass group from another mass group.
Indeed, ion isolation, molecular fragmentation, and mass analysis have been combined in a technique referred to as tandem mass spectrometry (or MS/MS) to thereby enhance identification of ion species. Tandem mass spectroscopy typically coupled with electrospray ionization (ESI) is a known technique utilized to produce gas phase ions of bio- and chemical molecules. Indeed, ESI is a soft ionization technique which produces multiply-charged molecular ions of large biomolecules. ESI continuously produces ions at normal atmospheric conditions. Once produced, the ions are introduced into a vacuum of a mass spectrometer using an atmospheric pressure interface. Liquid separation techniques such as for example high pressure liquid chromatography (HPLC), charge exchange (CE) generating radical ions of low internal energy, and on-line electrospray ionization mass spectrometry have all contributed to the success of modern biochemistry, pharmacology and health sciences. Even with these advances, the distinction of one large biomolecule from another depends on unique fragmentation patterns characteristic of the particular chemical bonding of the specific biomolecule.
Furthermore, the tandem mass spectrometer concept has been extended to triple quadrupole mass spectrometers. Triple quadrupole mass spectrometers have also been interfaced to electrospray ion sources. Triple quadrupole mass spectrometers offer medium resolution (up to several thousands Da) and low mass range (up to 2000-3000 Da) for MS/MS analysis. Further, systems known as QqTOF (or Q-TOF) combine two quadrupole mass sectors with time-of-flight mass analyzers (TOFMS). QqTOF techniques have been described for example by Morris et al, Rapid Commun. Mass Spectrometry, 1996, 10:889-896, and by Shevchenko et al, Rapid Commun. Mass Spectrom. 1997, 11:1015-1024, the entire contents of which are incorporated herein by reference. The QqTOF configuration can be considered as a replacement of the third quadrupole in a triple quadrupole instrument by a time-of-flight mass analyzer. The benefits of the QqTOF system are high sensitivity, mass resolution and mass accuracy in both precursor (MS) and product ion (MS/MS) modes. A particular advantage for full-scan sensitivity (over a wide mass range) is provided in both modes by the parallel detection feature available in TOF MS.
Fragmentation of ions is achieved in commercial tandem mass spectrometers through collisionally induced dissociation (CID) with buffer gas molecules in a quadrupole collision cell, see for example U.S. Pat. No. 6,285,027, the entire contents of which are incorporated herein by reference. In collisionally induced dissociation or fragmentation, the energy of collision is quickly redistributed over the large number of vibrational degrees of freedom available in large biomolecules. The energy redistribution leads to dissociation of bonds only of the lowest activation energy. Thus, the CID method seldomly provides sufficient MS/MS sequence information for proteins larger than 2 kDa. Since the excitation in CID is not specific, the most labile bonds are typically cleaved (which are often a modifying group) and not necessarily the structurally important bonds. Furthermore, CID requires the presence of the buffer gas at pressures of the order of 10 mTorr or more. Because the subsequent mass analyzer needs a relatively high vacuum for its operation, restricting apertures are introduced between the last fragmenting quadrupole and a second mass analyzer, thus reducing the number of ions transmitted and the overall sensitivity.
Electron capture dissociation (ECD) is a recent fragmentation technique that utilizes an ion-electron recombination reaction, as described by Zubarev et al, J. Am. Chem. Soc. 1998, 120: 3265-3266, the entire contents of which are incorporated herein by reference. The maximum cross section for the ion-electron recombination reaction occurs at very low electron energies (e.g., lower than 0.5 eV) and exceeds the collision cross section with neutral species by about 100 times. To date, ECD has been implemented in ion cyclotron resonance Fourier transform mass spectrometers (ICR-FTMS) with electrons injected directly into ICR cell only. Almost all ECD fragment ions come from a single bond cleavage. This makes electron capture dissociation well suited for protein sequencing. In contrast to CID, ECD is believed to be non-ergodic, i.e., the cleavage happens prior to any intramolecular energy redistribution. As a result, the ECD method cleaves more bonds than a conventional CID technique. Almost all known proteins in vivo contain post-translational modifications, which modulate and often define their biological function. Determination of the sites of these modifications is a top priority in proteomics studies. However, fragmentation using for example low-energy CID has the drawback of fragmenting the most labile bonds at the highest rate, which often are the linker bonds to the modifications. As a result, a modification group is often lost prior to the backbone fragmentation, making it difficult or impossible to determine a prior location of the modification group. In contrast, ECD cleaves specifically N—Cα bonds and imparts only a minimum of the internal energy into the fragments. The latter species, especially the even-electron c ions, i.e. one classification of fragmented peptides, retain the modification groups making their localization straightforward.
Recently, another type of ECD method referred to as “hot” electron capture dissociation (HECD) has been reported by Kjeldsen et al, Chem. Phys. Lett. 2002, 356: 201-206, the entire contents of which are incorporated herein by reference. Besides having a known maximum of electron capture dissociation (ECD) of gas-phase polypeptide polycations at low electron energy, a broad local maximum is found around 10 eV. The existence of this 10 eV maximum can be attributed to an electronic excitation prior to electron capture, a phenomenon similar to that in the dissociative recombination of small cations. In the HECD regime, not only N—Cα bonds are cleaved as in ECD, but secondary fragmentation is also induced due to the excess energy. Beneficially, this fragmentation includes abundant losses of, for example, CH(CH3)2 from Leucine and CH2CH3 from Isoleucine residues terminal to the cleavage site, which allows for distinguishing between these two isomeric residues. Even for larger molecules, the HECD produces abundant secondary fragmentation, despite the presence of substantially more degrees of freedom over which the excess energy could be distributed.
ECD/HECD ion fragmentation of biological molecules has been made in an ion cyclotron resonance Fourier transform mass spectrometer (ICR-FTMS) having electrons injected directly into the ICR cell. U.S. Patent Publication Application No. 2002/0175280, the entire contents of which are incorporated herein by reference, describes the use of electron capture dissociation for ion fragmentation in a three-dimensional ion trap. Since an electric potential inside the ion trap including the central point is time dependent, the electron source is kept at the highest positive potential achieved at the center of the ion trap during the RF cycle. Electrons can reach the ions stored inside the ion trap only during a period of few nanoseconds (or 0.1% of oscillation cycle) when the electric field potential at the center of the ion trap is close to the electron source potential. Together with a small size of aperture for electron beam introduction, this makes the effectiveness of ECD in this arrangement low.
Thus, to date, mass analyzers have not optimized electron capture dissociation to provide improved fragmentation and cleavage of input ionized species.